Diarrhea associated with inflammation or pathogen infection remains a major health problem in our veteran patient population with inflammatory bowel diseases (IBD) leading to frequent hospitalizations and increased healthcare costs. Therefore, a better understanding of the molecular pathophysiology of diarrhea is critically needed to develop effective and targeted therapeutic modalities. Diarrhea results from decreased intestinal absorption and/or increased secretion of fluid and electrolytes. A major route of electrolyte absorption in the human intestine involves coupled operation of Na+/H+ and Cl-/HCO3- exchangers. The gene Slc26a3, whose mutations are associated with congenital chloride diarrhea, encodes DRA (Down-Regulated in Adenoma), a protein known to play a critical role in mediating intestinal chloride absorption. Recent studies showing significant reduction in DRA expression in IBD suggest DRA to play key role in IBD-associated diarrhea and highlight DRA as a novel therapeutic target. Therefore, agents that activate DRA and/or counteract its downregulation may confer important antidiarrheal effects. In this regard, all-trans retinoic acid (ATRA), an active vitamin A metabolite, is known to improve intestinal epithelial integrity, exert anti-inflammatory and antidiarrheal effects. However, the mechanisms underlying antidiarrheal effects of ATRA are not known. Our current preliminary data and previous studies point towards bimodal pathway of ATRA effects on DRA involving (i) short-term non-genomic effects on stimulation of DRA function via increasing apical membrane DRA levels; (ii) long-term effects on DRA function via transcriptional activation of DRA expression. However, role of ATRA in counteracting reduced expression and/or altered membrane targeting of DRA in IBD- associated diarrhea is not known and merits in-depth investigation. Since optimal DRA function critically depends on its total cellular level as well as its apical membrane level, it is important to understand the cellular and molecular mechanisms that govern ATRA-mediated modulation of total cellular and apical cell surface expression of DRA in normal and inflammatory conditions. Therefore, we hypothesized that ATRA modulates DRA cell surface levels via distinct signaling and trafficking mechanisms. Since ATRA also upregulates DRA expression via transcriptional mechanisms, we further hypothesized that ATRA counteracts inflammation-associated diarrhea via modulating cellular expression and membrane targeting of DRA. The current application is, therefore, designed to investigate the cellular and molecular mechanisms of ATRA-mediated stimulation of DRA activity under normal and inflammatory conditions and evaluate the impact of upregulating DRA expression in response to ATRA or via transgenic overexpression in ameliorating inflammation and diarrhea in vivo as follows: Aim 1. Elucidate mechanisms of ATRA effects in regulating DRA membrane targeting under basal conditions and counteracting proinflammatory cytokine- induced decrease in DRA expression and membrane targeting; Aim 2. Establish the role of DRA in alleviating diarrhea in inflammatory mouse models via its ATRA-mediated upregulation or via transgenic overexpression of DRA and elucidate underlying mechanisms. We will utilize state-of-the-art approaches such as model human intestinal Caco-2 cells grown as three-dimensional cysts and human/mouse intestinal organoids that faithfully recapitulate the crypt-villus architecture of the intestinal epithelium to elucidate the mechanisms of ATRA-mediated modulation of cell surface DRA level. Anti-diarrheal potential of ATRA via upregulation of DRA will be evaluated in vivo utilizing mouse models of dextran sodium sulfate (DSS)-or IL-10 KO induced colitis. Role of DRA in alleviating IBD-associated diarrhea will be further verified in a transgenic mouse model of inducible intestine-specific overexpression of DRA. The outcome of our studies should enhance our mechanistic understanding of the importance of DRA as a therapeutic target for diarrhea and also unravel novel mechanisms underlying anti-diarrheal potential of ATRA, a dietary component.